Chemical Composition and Antiproliferative and Antioxidant Activities of Essential Oil from Juniperus phoenicea L. Cupressaceae

 

Hana R. Bajes¹*, Sawsan A. Oran², Yasser K. Bustanji³ 

^1,2 Department of Biological Sciences, The University of Jordan, Amman, Jordan.

³School of Pharmacy, The University of Jordan, Amman, Jordan.

³Hamdi Mango Center for Academic Research, The University of Jordan, Amman, Jordan.

 

ABSTRACT:

Being multipurpose, relatively safe, and widely favorable for consumption, interest in the essential oils of medicinal plants has been increasing. Essential oil (EO) of Juniperus phoenicea is traditionally used for treatment of several health problems such as diabetes, rheumatism, and cancer that is the second leading cause of death in Jordan. This study aims to collect and chemically analyze EO from Juniperus phoenicea L., from Jordan and to evaluate its cytotoxic and antioxidant activity against human breast cancer cells (T47D), colorectal adenocarcinoma cells (CACO2), and normal human fibroblasts (MRC5). EO was extracted by hydro-distillation and analyzed in a gas chromatograph coupled with a mass spectrometer. Cell viability was assessed using trypan blue, neutral red, and MTT assays, and antioxidant activity was evaluated using DPPH scavenging activity assay. Chemical composition analysis revealed 23 constituents in the EO, and the amount of α- pinene was the highest (69.71%). The results also revealed that the IC50 values of the viability assays were higher among normal cells compared to the human cancer cell lines, and the viability inhibition was significant at higher concentrations compared to untreated cells. Nevertheless, low antioxidant activity was observed for the oil in the DPPH scavenging activity test. To sum up, this study indicates that Jordanian Juniperus phoenicea EO, albeit unlikely to be an effective antioxidant, is optimistically potential to be utilized in breast and colon cancers treatment due to its preferential cytotoxicity against cancer cells.

 

 

 

INTRODUCTION:

The various phytogeographical areas of Jordan are rich in a variety of wild plants¹. Twenty percent of all Jordan’s plant species are medicinal plants that are used by local inhabitants and in pharmaceutical industry². There are more than 2500 plant species, 868 genera, and 142 families^{1, 3-5}. Medicinal plants that are rich in bioactive compounds are abundant in Jordan’s geographic regions. In Jordan, more than 485 species of medicinal plants and 99 families have been identified as therapeutic agents⁶. Medicinal plants in Jordan can be exemplified by Varthemia iphinoides, Bongardia chrysogonum, Ajuga chia, Salvia palaestina, Micromeria nervosa and Rubus sanguineus ^{2, 3, 7, 8}.

 

There has been increasing attention in the essential oils (EOs) of medicinal plants because of their ability to be utilized for several purposes, especially as they are relatively safe and widely accepted by consumers^{9, 10}. It is urgent and essential to discover natural and plant -based products that can prevent the growth of cancer cells with minimum side effects¹¹. EOs are secondary metabolites isolated from aromatic plants, with volatile and complex chemical structure that mainly contains terpenes as well as some other non-terpene constituents. A wide variety of free radical scavenging molecules are found in plants, such as, carotenoids, vitamins, dietary glutathione, flavonoids, and anthocyanins are rich in antioxidant activities¹².

 

A variety of plant families in Jordan has been investigated for their anti-tumoral effect^13-17. In Jordan, cancer is the second leading cause of death after heart disease, and breast and colon cancers are among the most prevalent types¹⁸.

Plants of the genus Juniperus have been used as a spice, flavoring for alcoholic drinks, as well as in cosmetics¹⁹, and they are traditionally used by Jordanians as an important medicinal plant in the treatment of diabetes, diarrhea, bronco-pulmonary disease, and rheumatism²⁰. In South Jordan, decoction of J. phoenicea leaves is widely used and it is recommended for treatment of rheumatism. Also, the aqueous extract of J. phoenicea leaves collected in South Jordan exhibited a significant antidiarrheal effect by reducing intestinal fluid accumulation and inhibiting intestinal motility, while the ethanolic extract showed potential antifertility effects in male albino rats^{3, 21}. Juniperus phoenicea L. is an evergreen shrub or tree widely distributed on the Mediterranean basin. As a tree, it can reach a height of 10 m, with berries about 1 cm in diameter and composed of 6–8 scales with 3–9 seeds^{1, 20, 24}.

 

Research on J. phoenicea showed antioxidant^{20, 23-30}, antidiabetic²⁵, ulcer protective²³, and antimicrobial activities^{20, 24, 31}. Scavenging of the reactive oxygen species (ROS) in the body, such as hydroxyl radical, hydrogen peroxide superoxide anion, and singlet oxygen, which may lead to DNA damage, is due to the presence of phytochemical constituents present in plants³².

 

Some studies reported the cytotoxic activity demonstrated by J. phoenicea against human cancer cell lines. Oils from berries and leaves of J. phoenicea showed very high cytotoxic potential against human brain, cervix, lung, liver, and breast cell lines³¹. Methanolic extracts from the leaves of the plant demonstrated cytotoxic potential against human bladder, liver hepatocellular, lung, and breast carcinomas³³. A study showed that the ethanolic extract of J. phoenicea twigs and leaves were cytotoxic in human cervix carcinoma³⁴. A methanolic extract of J. phoenicea from Saudi Arabia and Indonesia displayed high cytotoxicity in human laryngeal carcinoma cell lines³⁵. In addition, this plant demonstrated cytotoxicity effect against both human breast adenocarcinoma and colon carcinoma cell lines³⁶.

 

In this context, J. phoenicea is selected to be studied based on the existing preliminary data that suggest its potential for cancer treatment and balancing oxidative status. To the best of our knowledge, this study is the first to evaluate the antioxidant activity as well as the cytotoxic potential of EOs extracted from J. phoenicea from Jordan.

 

MATERIALS and METHODS:

Plant material:

Aerial parts of J. phoenicea (leaves and berries) were collected from Petra area (30°20'48.8"N+35°28'08.0"E) in south east Jordan during February 2020. The plants were taxonomically and authentically identified by Professor Sawsan Oran; a plant taxonomist, (School of Biological Sciences, the University of Jordan). Voucher specimens have been deposited at the herbarium of the Department of Biological Sciences- University of Jordan [specimen 2].

 

EO extraction:

1200 g of the collected plant was dried in shade at room temperature and ground, and Clevenger apparatus was used to isolate EO from the plants by hydro-distillation 4 hrs. The percent yield was calculated as weight per weight (W/W) based on the dry weight of plant material³⁷. The water content in EO was dried by anhydrous sodium sulphate and stored in dark glass vials at 4°C³⁸.

 

EO chemical analysis:

Gas chromatography (GC) and mass spectrometry (MS) methods were used to find the phytochemical composition of the EO. 0.1 µl of the EO was injected in the TRACE GC 2000 SERIES quantitative gas chromatograph equipped with a split-splitless injector, helium was the carrier gas at a flow rate of 0.1ml/min. Compounds identification was conducted by matching their spectra with the Wiley and NIST libraries in the software as well as with the retention index values reported in Adam’s Library³⁹.

 

Cell culture:

Human colorectal adenocarcinoma cells (CACO2), breast cancer cells (T47D), and normal human fibroblasts (MRC5) were purchased from American Type Culture Collection company (ATCC). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) mixed with 10% fetal bovine serum (FBS), 1% L-glutamine and penicillin (100 U/ml) and incubated in a humidified atmosphere of 5% CO2 at 37°C. After three days of incubation the cells were harvested by trypsin-EDTA solution and incubated for 10 minutes. 3 ml of culture media was added to the flask, and then the cells were transferred to a tube and vortexed. Finally, the cells were counted, and viability test was performed by trypan blue dye test. The cells were seeded in multi-well plates at the appropriate density recommended for each test^{38, 40}.

 

Cytotoxicity Assays:

Trypan exclusion assay:

Trypan blue assay is one of the earliest and most common methods for counting cells and determining increase or decrease in number of viable cells to indicate proliferation activity⁴¹. Viable cells with intact plasma membranes that are not destructed by the EO remain clear and exclude the blue dye, while nonviable cells with ruptured membrane, regardless of death mechanism, are stained blue under the light microscope⁴². Cells were seeded at high concentration (1.0 x10⁴ cells/ml) in 96-well plates and incubated at 37 °C to allow for cell attachment. Then 100 ul of drug mixed with media was added to each well and allowed to incubate for 72 hours. After 72 hours, the contents of the wells were emptied by trypsinization and 25 μl of the cell suspension and 25ul of trypan blue dye (sigma) were mixed in a mixing well. After 30 sec., 25 ul of the mixture was added to a hemocytometer and observed under microscope. The number of stained cells and the total number of cells were counted. The IC50 indicating a net loss of cells following treatment was calculated from:

[(Ti-Tz)/Tz] x 100 = – 50, where Ti is the initial cell count and Tz is the final cell count⁴¹.

 

Neutral Red Assay:

Neutral red assay is one the most common employed tests for the detection of cell viability following exposure to anticancer substances. The assay is based on detecting the accumulation of the neutral red dye in the lysosomes of viable, uninjured cells⁴³. According to previous procedure⁴⁴, about 10, 000 cells from each cell line were seeded in each well of 96-well plates and incubated overnight at 37°C and 5% CO2. The J. phoenicea EO was dissolved in 100 µl of 5% DMSO and added to the cells at final concentrations of (800, 400, 200, 100, 50) µg/mL, and incubated again for 24 hours. Doxorubicin was used as positive control. Then, the cells were washed with phosphate-buffered saline (PBS) and the supernatant was discarded. A total of 100 μl neutral red (NR) solution (50 μg/ml) was added and incubated at 37°C for 2 hours. NR then was removed, and wells were washed with PBS, and after 10 minutes, absorbance was detected by a dual-wavelength UV spectrometer at 520 nm with a 650 nm reference wavelength. The percentage of antiproliferative activity compared to the untreated cells was determined as: % Antiproliferative activity = [100×(Absorbance of untreated group−Absorbance of treated group)]/Absorbance of untreated group.

 

MTT cytotoxicity assay:

EO of J. phoenicea was diluted in 5% Dimethyl Sulfoxide (DMSO) solution and added to about 10, 000 cells from each cell line (T47D, CACO2, and MRC5) at final concentrations of 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 µg/ml, and the cells were incubated for 72hrs. After exposure, 3-(4, 5-dimethylthiazol-2w-yl)-2, 5-diphenyl tetrazolium bromide (MTT) was added to the wells and incubated for another 4 hours. 100 ul of DMSO solution was then added to solubilize the MTT crystals before reading optical density (OD) by multi-well plate reader (Bio-Tek Instrument, USA) at 570 nm using a reference wavelength of 630 nm^{38.40.45.46.11}. Additionally, doxorubicin at final concentrations of 0.1, 0.5, 1, 5, 10, 25, 50, 100, 200 µg/mL was used as positive control. The percentage of viability was calculated as the absorbance ratio between treated and untreated (control) wells; hence the absorbance of untreated cells was considered as 100%. Percentage of growth inhibition of cells was calculated as follows: % Inhibition = 100 − (Treated OD/Non-treated OD) × 100)⁴⁷. IC50 values were calculated as concentrations that exhibited 50% inhibition of proliferation on the tested cell line.

 

Antioxidant activity:

Following a published protocol⁴⁸, the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity assay was employed to measure the antioxidant activity. DPPH is useful to evaluate scavenging activity of specific compounds, by reacting with substance able to donate a hydrogen atom to become stable^{49, 50}. Briefly, a volume of 200 mL of 400, 600, 800, and 1000 µg/ml of EO or ascorbic acid (standard) were prepared in 95% ethanol and added to 2 mL DPPH (0.21 mM in 95% ethanol). The mixture was shaken, left for 60 min at room temperature in the dark, and the absorbance was detected at 517 nm in a spectrophotometer. The percentage of DPPH inhibition were calculated using the following equation: percentage of inhibition = [(Ac -As)/(Ac)] x 100, where Ac is the absorbance of the control reaction and As is the absorbance of the sample reaction. The sample concentration (in 1 mL reaction mixture) providing 50% inhibition (IC50) was estimated by plotting percentages of inhibition against concentrations of sample.

 

Statistical analysis:

All assays were conducted in triplicate. The results were expressed as mean ± SD, and the differences between the means were tested for significance in the statistical analysis software IBM SPSS Statistics Version 23 (Armonk, New York, USA) using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. The significance level was set at P < 0.05.

 

RESULTS AND DISSCUSSSION:

J. phoenicea EO composition:

Table 1 shows that 23 components were identified in the EO of J. phoenicea, amounting for 99.11 % of the total content, and the percent yield of the EO was 1.0% (w/w). The EO was found to be rich in monoterpene hydrocarbons (77.99 %), 13.86 % of the content were oxygenated monoterpenes, and sesquiterpene hydrocarbons were the least abundant (7.10 %).

 

The analysis showed that α- pinene is the main component identified in J. phoenicea EO (69.71 %). The other major identified compounds were italicene epoxide (13.63 %), and sylvestrene (3.84 %). A similar study conducted in Algeria found 51 compounds, and the analysis quantified α- pinene to be the most abundant (40%)⁵¹. All the identified compounds are types of terpenes.

 

The major identified monoterpenes were α- pinene and sylvestrene. The major oxygenated monoterpene identified was italicene epoxide. β- Cedrene was the major sesquiterpene identified (2.71 %). On the other hand, the mass spectra of some peaks spiking the underlying broadened unresolved band (Rt= 19.738, Rt= 33.074) suggested the presence of monoterpenes and sesquiterpenes referred to as unknown in Table 1.

 

In previous studies, chemical analysis of EO samples from J. phoenicea revealed the prevalent presence of α-pinene, sabinene, monoterpenoids, sesquiterpenoids, α -cedrol, δ-3-carene, limonene, germacrenes, and γ -cadinene ^{20, 25, 27, 31}. Similar to the findings of the current study, three earlier studies found α-pinene the major compound in the EO of J. phoenicea, with concentrations of 39.30%³¹, 55.7%²⁰, and 24.02%²⁵.

 

Table 1: Chemical composition of EO hydro-distilled from the leaves of Jordanian J. phoenicea analyzed by GC-MS.

 

Compounds are listed in order of their elution times from a DP-5ms column; ^a Experimental RI relative to (C₈-C₂₀) n-alkanes; ^b Literature RI based on reference³⁹; ^c the percent content is based on the compound relative peak area; ^d unidentified compound; ^e value not available in literature; compounds in bold are the major (≥ 4%); ^f the value of unknown compound was not included in the total.

Cytotoxicity assays:

In order to evaluate the antitumoral effect of J. phoenicea EO on the two studied cancer cell lines (Caco II and T47D) and fibroblasts, three in vitro cytotoxicity/ cell viability assays, one dye exclusion test (trypan blue) and two colorimetric methods (MTT and neutral red), were applied on the cells.

 

Table 2 illustrates the IC50 values calculated for the tested EO against the three cell lines. Also, doxorubicin’s IC50 values were calculated from MTT test and they ranged between 2.66 and 6.3 µg/ml, which are consistent with the normal values reported in the literature for this cytotoxic drug^52-54, and that confirms the accuracy of the experiments.

 

Table 2: IC₅₀ values (mean ± SD µg/ml) of J. phoenicea EO from three cytotoxicity assays.

 

The results obtained from trypan blue assay confirm the preferential cytotoxicity of the EO on cancer cells compared to normal fibroblasts; IC50 was 249.6 ± 0.26 µg/ml for Caco II cells and 544.6 ± 0.44 µg/ml for T47D cells, compared to 665.2 ± 0.21 µg/ml in the case of the Fibroblasts (MRC5). With regards to the neutral red and MTT assays, the table also shows that the EO demonstrated higher inhibitory effect on the growth of the cancer cell lines, compared to the normal cells; neutral red values of IC50 were 244.7 ± 0.88, 330.3 ± 1.10, and 492.7 ± 1.41 µg/ml for Caco II, T47D and MRC5 cell lines, respectively, and the values from MTT assay were 476.3 ± 0.43, 244.7 ± 0.26, and 544.6 ± 0.43 µg/ml.

 

The difference in IC50 values between tumoral and normal cells suggests the potential of EO to be used in treatment of cancer. Moreover, according to the classification of natural constituents cytotoxicity, that describes ingredients with IC50 values of 100 – 1000 µg/ml as potentially harmful to cancerous cells⁵⁵, our cytotoxicity results indicate the possibility of considering J. phoenicea EO as a healing suggestion in cancer treatment.

 

The significance of the differences between the mean percentages of the viability according to EO concentration is illustrated in Fig. 1 and Fig. 2 for the results of MTT and neutral red assays, respectively. For all the studied cell lines, Fig. 1 indicates significantly lower viability mean values recorded for the highest concentration of EO. Similarly, as shown in Fig. 2, neutral red results illustrate that the EO inhibited the growth of the three cell lines on a dose dependent manner; compared to the control group, treated cells showed significant decrease in viability as the dose of EO increased.

 

The cytotoxicity of J. phoenicea EO could be explained by the high content of total monoterpenes, α-pinene and β-pinene, as explained by El-Sawi and her team³¹. Interestingly, Al Groshi and her colleagues have reported cytotoxic activities by terpenes of J. phoenicea against human cancer cell lines³³. Additionally, caryophyllene C, which was found in a percentage of 0.64%, was reported to have mild sedative properties with an in-vitro cytotoxic activity against breast cancer cells⁵⁶.

 

Fig. 1: The effect of different concentrations of J. phoenicea EO on the viability of Caco II (A), T47D (B), and MRC5 (C) cell lines, by MTT assay. Bars represent means and standard errors. *p < 0.01, in comparison to control.

Those findings suggest that terpenes played an important role in inducing cytotoxicity in our experiments. El-Sawi’s study³¹ was conducted on plants of J. phoenicea EO from Egypt, and resulted in IC50 values for the EO on CACO II and T47D cells lower than our numbers. However, with regards to Jordanian J. phoenicea EO, no cytotoxicity data could be found in the literature to compare our results with. In accordance with our findings, relatively high IC50 values were obtained for J. scopulorum and J. horizontalis EOs, 278.8 µg/ml and 245.5 µg/ml, respectively⁵⁷. As opposed to EOs, the majority of the researched literature have shown lower IC50 values when ethanolic, methanolic or infusion extracts were applied on cancer cell lines^33-36.

 

Fig. 2: The effect of different concentrations of J. phoenicea EO on the viability of Caco II (A), T47D (B), and MRC5 (C) cell lines, by Neutral Red assay. Bars represent means and standard errors. *p < 0.01, in comparison to control.

 

Antioxidant activity:

The antioxidant activity of J. phoenicea EO is expressed in Table 3 in terms of IC50 values. The IC50 value for the DPPH radical scavenging activity was 897.8 ± 0.35 µg/ml.

 

Table 3: Antioxidant activity of J. phoenicea EO (Mean ± SD µg/ml).

 

Several studies have described the antioxidant activity of J. phoenicea^{20, 23-30}. The low antioxidant activity shown in this study is potentially attributed to the high content of terpene hydrocarbons in the EO²⁰; 77.99 and 7.10 % of the EO’s content in our experiments were monoterpene hydrocarbons and sesquiterpene hydrocarbons, respectively. It was previously concluded that the environment of higher polarity allows for higher capacity of radical scavenging²⁴. The observed activity maybe was dependent on the oil’s major constituent; α-pinene (69.71%), despite it was reported in other studies to be a strong antioxidant⁵⁸.

 

CONCLUSION:

To our knowledge, this is the first study for the chemical analysis the EO contents of J. phoenicea and the evaluation the antiproliferative of antioxidant potential for the Jordanian species J. phoenicea, although it has been studied in other countries.

 

The EO of J. phoenicea from Jordan was found to be rich in monoterpene hydrocarbons and α- Pinene was the predominant component. The biological active compounds explain preferential in-vitro antiproliferative potential of the EO on the tested cancer cell lines. J. phoeniceae is recommended for further studies and identification of the main phytochemical components and their anti-proliferative mechanism.

 

FUNDING INFORMATION:

This project was funded by the Deanship of academic research of the University of Jordan- Amman.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 27.04.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):153-159.